Fat-Soluble Vitamins Analysis on anAgilent ZORBAX Eclipse PAHPolymeric C18 Bonded Column

Abstract

Fat-soluble vitamins are highly lipophilic molecules which are analyzed by normal

phase HPLC or reversed phase HPLC with a methanol/acetonitrile mobile phase. A

new reversed phase HPLC method was developed for the Agilent ZORBAX Eclipse

PAH column for vitamins A, D2, D3 and E. This polymeric C18 phase column provides

baseline separation of vitamins D2 and D3, with a short analysis time of only three

minutes. The methanol and water mobile phase made this compatible with

LC/MS/MS with electrospray ionization.

Authors

Rongjie FU

Agilent Technologies (Shanghai) Co. Ltd.

412 Ying Lun Road

Waigaoqiao Free Trade Zone

Shanghai 200131

P.R. China

Jianzhong LI and Ying Wang

Agilent Technology Inc.

P.R. China

Application NoteFood

2

Introduction

Vitamins are essential nutrients that are necessary in smallamounts for various functions in the human body. Vitaminsare divided into two groups: water-soluble (such as B-com-plex and C vitamins) and fat-soluble (such as vitamins A, D, Eand K). Fat-soluble vitamins perform many functions in thebody. Vitamin A plays an important role in helping the eyesadjust to light changes, bone growth, tooth development,reproduction, cell division and gene expression. Vitamin Dhelps the body to use calcium and phosphorous, and itincreases the amount of calcium absorbed from the smallintestine to help form and maintain bones. Children especiallyneed adequate amounts of vitamin D to develop strong bonesand healthy teeth. Vitamin E acts as an antioxidant, protectingvitamins A and C, red blood cells and essential fatty acidsfrom destruction. [1] Though fat-soluble vitamins are benefi-cial for human health, they have a toxic effect if taken at veryhigh levels. Therefore, the quantity of fat-soluble vitamins infortified foods must be controlled.

HPLC methods are usually used to analyze for these vitamins.Vitamins A and D are often analyzed by normal phase LC asdescribed in the USP method for vitamin A and D. [2] VitaminD has different forms; vitamins D2 and D3 are the most impor-tant compounds. They have similar structures that make themdifficult to separate by HPLC. Recent research showed vita-min D2 is much less effective than vitamin D3 in humans. [3]Therefore, most foods fortified with Vitamin D use cholecal-ciferol (vitamin D3). Some analytical methods for Vitamin D infood use ergocalciferol (Vitamin D2) as an internal standard(for example, The Association of Official Analytical ChemistsInternational (AOACI) method 2002.05: cholecalciferol (vita-min D3) in selected foods, such as milk and cheese).Therefore, it is important that any method used is capable ofaccurately separating vitamins D2 and D3. [4]

This application note describes a method for simultaneouslyanalyzing vitamins A, D2, D3 and E on a polymeric bondingC18 phase. This C18 phase gives the selectivity necessary forpolynuclear aromatic hydrocarbons PAH’s, some of whichseparate on the basis of shape. The selectivity of this phasecompared to that of typical monomeric bonding C18 phasesmakes it ideal for separating vitamins D2 and D3, using the

same principle of shape selectivity but with a shorter analysistime. The simple mobile phase of methanol and water is alsoideal for LC/MS with electrospray ionization (ESI).

Experimental

HPLC conditions:

Instrument: Agilent 1200SL series LC system with binary pump

Column: Agilent ZORBAX Rapid Resolution HT Eclipse PAH, 4.6 mm × 50 mm, 1.8 µm

Temperature: 40 °C

Mobile phase: 92% methanol/8% water

Flow rate: 2 ml/min (0.6 ml/min for MS detector)

Wavelength: 325 nm for VA/280 nm for VD and VE

Injection: 2 µL

Standards: Vitamin A, ergocalciferol (vitamin D2), cholecalciferol (vitamin D3) and Vitamin E in methanol

Mass conditions:

Mass instrument: Agilent 6460 triple quadrupole LC/MS system

Ion source: ESI

Polarity: Postive/Negative

Source temp: 325 °C

Gas flow: 5 L/min

Nebulizer: 55 psi

Sheath gas temp: 400 °C

Sheath gas flow: 12 L/min

Nozzle voltage: Negative 1000 V; Positive 0 V

Ion spray voltage: 4000 V

Resolution: Q1 (unit) Q3 (unit)

Scan mode: Multiple Reaction Monitoring (MRM)

Compound Precursor Product Collisionname ion ion Fragmentor Energy Polarity

Vitamin A 269.2 213.2 110 7 PositiveVitamin D2 397.2 379.4 120 4 PositiveVitamin D3 385.3 367.3 120 5 PositiveVitamin E 429.3 163.1 150 25 Negative

Delta EMV: 200 V

3

Results and Discussion

Vitamins A, D2 and D3 are usually separated with normalphase columns due to the highly lipophilic properties of thesemolecules. The methods developed on reversed phasecolumns generally use methanol/acetonitrile as the mobilephase, but mostly do not baseline resolve Vitamins D2 andD3. A previous application was developed for fat-soluble vita-mins on Agilent ZORBAX Eclipse XDB C18 and Agilent ZORBAX StableBond SB-C18 columns, but with lower resolu-tion of vitamins D2 and D3 [5]. A new application comparedthe separation of vitamins D2 and D3 with Stablebond C18bonded phase to the same separation with Eclipse Plus C18bonded phase. The SB-C18 may resolve the D vitamins better

because of the accessible silanols on the surface of the sta-tionary phase. However, the longer column increases the res-olution but also increases the analysis time. The flow rate canbe raised to decrease the time. The method was developed asa UHPLC method for high pressure exceeding 600 bar [4].

The results of this study determined that a fat-soluble vita-mins analysis on an Eclipse PAH column provides better reso-lution and shorter analysis time compared to Eclipse PlusC18. Vitamins D2 and D3 were baseline separated in 3 min-utes (Figure 1). The polymeric bonding C18 phase made iteasy for D2 and D3 to resolve completely. The pressure wasonly 268 bar with a 2 mL/min flow rate, which can be per-formed on any LC instrument.

30

25

20

15

10

5

0

_5

1 2

1

23

4

1

2 3 4

3 4 5 6 7 8 9 min

30

25

20

15

10

5

0

_5

1 2 3 4 5 6 7 8 9 min

1 Vitamin A2 Vitamin D23 Vitamin D34 Vitamin E (a-VE)

Agilent ZORBAX Rapid Resolution HTEclipse Plus C18 4.6 mm × 50 mm, 1.8 µm

Agilent ZORBAX Rapid Resolution HTEclipse PAH 4.6 mm × 50 mm, 1.8 µm

Rs 2,3 = 1.60

Figure 1. Chromatograms for the separation of four fat-soluble vitamins on different Agilent ZORBAX C18 columns.

4

Figures 2 and 3 show the total ion chromatogram (TIC) andmultiple reaction monitoring (MRM) of four vitamins on theZORBAX Eclipse PAH column. All four compounds were wellresolved with no ion depression, which made it possible toget very high MS sensitivity.

VA

VD2

VD3

VE

7.5

×103

7

6.5

6

5.5

5

4.5

4

3.5

3

2.5

2

1.5

1

0.5

0.5 1 1.5 2 2.5 3 3.5 4 4.5

Counts vs. acquisition time (min)

5 5.5 6 6.5 7 7.5 8 8.5

Figure 2. TIC of four fat-soluble vitamins on an Agilent ZORBAX Eclipse PAH column.

5

×103

3

2

1

5.28

0.5 1 1.5 2 2.5 3 3.5 4 4.5

Counts vs. acquisition time (min)

5 5.5 6 6.5 7 7.5 8 8.5

×103

3

4

2

1

1.41

0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5

×103

4

6

2

4.78

0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5

×103

1

1.5

0.5

4.34

0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5

Figure 3. MRM of four fat-soluble vitamins on Agilent ZORBAX Eclipse PAH column.

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Agilent shall not be liable for errors contained herein orfor incidental or consequential damages in connectionwith the furnishing, performance, or use of this material.

Information, descriptions, and specifications in this publication are subject to change without notice.

© Agilent Technologies, Inc., 2010Printed in the USAFebruary 15, 20105990-5342EN

Conclusion

The unique selectivity of a polymerically bonded C18 column,such as the Agilent ZORBAX Eclipse PAH column makes itpossible to separate compounds with very similar structureslike vitamins D2 and D3. The method developed in this appli-cation note resolved four fat-soluble vitamins simultaneously,in 3 minutes, and is compatible with the MS detector. Themethod can be run on any LC instrument and is suitable forthe analysis of fat-soluble vitamins in foods.

References

1. http://www.ext.colostate.edu/pubs/foodnut/09315.html.

2. Fat- and Water-Soluble Vitamins with Minerals Tablets,USP30-NF25.

3. A. Laura, G. Armas, Bruce W. Hollis, and Robert P.Heaney, Vitamin D2 Is Much Less Effective than VitaminD3 in Humans. The Journal of Clinical Endocrinology &Metabolism 89(11):5387–5391.

4. John W Henderson Jr and Judy Berry, “UHPLC MethodDevelopment Options for a Vitamin D2 and D3Separation,” Agilent Technologies publication 5990-5091EN.

5. R. Ricker, "Comparison of Non-Endcapped StericallyProtected Silanes to Fully Endcapped Dimethylsilane,"Hewlett Packard Lab Insights, FD15, 1998.

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